JPH07242688A - New physiologically active substances nk148198a and nk 148198b, their production and use - Google Patents

Abstract

PURPOSE:To obtain the subject new physiologically active substances, having suppressing actions on the growth of a mammalian cell and useful as an active ingredient of an anticancer agent by culturing a microorganism, belonging to the genus Streptomyces and having the ability to produce the physiologically active substances NKl48198A and NK148198B. CONSTITUTION:This method for producing new physiologically active substances, NK148198A and NK148198B is to culture a microorganism, belonging to the genus Streptomyces and having the ability to produce the physiologically active substance, NK148198A or NK148198B [e.g. Streptomyces sp. NK148198 strain (FERM P-13962)] in a culture medium, produce and accumulate the formed product in the resultant culture and collect the prepared product. The physiologically active substances assume a white waxy appearance, are soluble in lower alcohols, chloroform and ethyl acetate and manifest the positivity to color reactions such as phosphomolybdic acid- sulfuric acid and ninhydrin reactions. Furthermore, the resultant physiologically active substances comprise the NK148198A expressed by the rational formula, C14H31N and the NK148198B expressed by the rational formula, C15H33N and are useful as an active ingredient of an anticancer agent.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel physiologically active substance NK
The present invention relates to a method for producing 148198A or NK148198B and its use. The compound of the present invention and a salt thereof have a cell growth inhibitory action and are expected as physiologically active substances used as chemotherapeutic agents for malignant tumors.

[0002]

Conventionally, as anticancer agents, adriamycin, bleomycin, cisplatin, etc. have been known.

[0003]

However, these are highly toxic and are not satisfactory. The invention of new compounds suitable for these uses is awaited.

[0004]

Therefore, the present inventors have
As a result of various searches for microbial metabolites, one strain belonging to the radiant bacterium has physiologically active substances NK148198A and NK1481 which have a growth inhibitory action on mammalian cells.
It was found to produce 98B. More specifically, it relates to an anticancer substance containing the compound and a pharmacologically acceptable salt thereof as an active ingredient. The compound forms a salt with an acid. As the salt for forming the salt, for example, a pharmacologically acceptable acid may be used, and for example, hydrochloric acid, sulfuric acid, phosphoric acid and the like are preferable.

The present invention has been completed based on the above findings. The above physiologically active substance NK148198A or NK148198B is N belonging to the genus Streptomyces.
It can be obtained by culturing a K148198A- or NK148198B-producing bacterium, allowing the compound to be produced and accumulated, and collecting from this culture. NK148198A or NK
As a representative 148198B-producing bacterium, Streptomyces sp. NK14819 isolated from soil was used.
8 strains (Ministry of Industrial Research, No. 13962).

The mycological properties of the NK148198 strain are shown below. 1. Morphological properties As a result of observing after 2 weeks at 27 ° C, the aerial hyphae are simply branched, and their tips are loop-shaped or spiral-shaped. No formation of sporangia or uniaxial raw silk is observed. No zoospores are allowed. The spore surface is smooth or rough, the spores are cylindrical, and the size is 0.6 to 0.8 x 0.8 to 1.
2 μm. In addition, spores are formed by forming a chain of 10 or more.

2. Growth on various media Table 1 below shows the growth conditions on various media at 27 ° C for 2 weeks.

[0008]

[Table 1]

3. Physiological properties 1. Suitable growth range: 24-32 ° C 2. Reduction of nitrate: Positive 3. Liquefaction of gelatin (on glucose / peptone / gelatin medium at 20 ° C): Positive 4. Hydrolysis of starch (starch / inorganic salt agar)
: Positive 5. Coagulation of skim milk: Negative 6. Peptonization of skim milk: Positive 7. Formation of melanin-like pigment: Positive 4. Utilization of carbon source (on Pridham Godleybe agar medium) L-arabinose + D-xylol-D-glucose + D-fructose-sucrose + inositol-L-rhamnose-raffinose-D-mannitol-

5. Diaminopimelic acid in the cell wall LL-diaminopimelic acid. To summarize the above,
The cell wall of this strain is LL-diaminopimelic acid, and according to the method of the International Streptomyces genus project (abbreviated as ISP), the morphology of sporulating mycelia belongs to Section Spirals (Spairales) and the spore surface is smooth or Rough, mature hyphae are red color-series, producing melanin-like pigments,
It exhibits a yellow to brown pigment in the medium. Further, the color of the basal hyphae is pale yellow or pale brown. L as a carbon source
-Arabinose, D-glucose and sucrose are used, and D-fructose, D-xylose, inositol, L-rhamnose, raffinose and D-mannitol are not used.

Based on the above properties, Bargeys Manual of Deterrainative Bacteriolo (Bergey's Manual of Deterrainative Bacteriolo), edited by R. E. Buffanan & N. Gibbons
gy) 8th edition, 1974, and as a result, it was found that the above NK148198 strain belongs to the genus Streptomyces. Therefore, this bacterium was designated as Streptomyces sp. NK148198. The strain is a product of the Institute of Biotechnology, Institute of Industrial Science and Technology,
Deposited as FERM P-13962.

The strains belonging to the genus Streptomyces used in the present invention, like other strains of the genus Streptomyces, are likely to change their properties, and are easily mutated by artificial mutation means using, for example, ultraviolet rays, X-rays and chemicals. And any kind of mutant strain, the physiologically active substance NK148198A or (and) N, which is a target of the present invention, can be used.
K148198B (including both below, simply NK1481
Those having a production capacity of 98) can be used in the present invention.

To produce NK148198 according to the present invention, the strain is first aerobically cultivated in a medium containing nutrients that actinomycetes can utilize. As the nutrient source, known ones conventionally used for culturing mold can be used,
For example, as the carbon source, glucose, fructose, glycerin, sucrose, dextrin, galactose, organic acid and the like can be used alone or in combination.

As the inorganic and organic nitrogen sources, ammonium chloride, ammonium sulfate, urea, ammonium nitrate,
Sodium nitrate, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean flour, cottonseed oil residue, casamino acid, bacto soyton, soluble vegetable brotin, oatmeal and the like can be used alone or in combination.

In addition, inorganic salts such as sodium chloride, calcium carbonate, magnesium sulfate, copper sulfate, iron sulfate, zinc sulfate, manganese chloride, and phosphate can be added, if necessary, and organic substances such as amino acids, vitamins and nucleic acids. It is possible to appropriately add a substance or an inorganic substance. The most suitable culture method is a liquid culture method, especially a deep agitation culture method.
The culture temperature is preferably 15 to 35 ° C., and the pH is preferably neutral to slightly acidic.

In the liquid culture, when the culture is generally carried out for 3 to 5 days, the substance NK148198 is produced and accumulated in the culture medium. When the production amount in the cultured cells reaches the maximum, the culture is stopped,
The cells are separated from the culture medium by filtration, and the desired product is purified and isolated. For the purification and isolation of this substance from the filtrate, generally, the method of separation and purification used for the isolation of microbial metabolic products from cultured cells is used. The purified product thus obtained is NK14819.
Since it is a mixture of 8A and NK148198B, it is chemically modified and then separated and purified. Finally, by chemically removing the modifying group, NK148198A and NK148198B can be produced.

That is, the culture solution is separated into bacterial cells by a usual filtration method. The obtained cells were extracted with methanol and then concentrated under reduced pressure to dryness. After extraction with butanol, chloroform: methanol silica gel column, LH-2
0, Sephadex column, butanol: Aqueous-based silica gel column, sequentially applied, and NK148198A and N
A mixture of K148198B is obtained. Then, this is converted into a paramethoxybenzyloxycarbonyl compound (PMZ compound),
After chemical conversion, NK1 was applied to reverse phase HPLC.
PMZ of 48198A and NK148198B respectively
The bodies were separated and sorted. Finally, by chemically removing the PMZ group, NK148198A and NK148
198B is obtained.

The physiologically active substance N obtained as described above
The physicochemical properties of K148198A and NK148198B are shown below. a) NK148198A 1) Appearance; white wax 2) Molecular weight; HR-FAMS (m / z): 214.2532
4 (M + H) ⁺ 3) Rational formula; C ₁₄ H ₃₁ N 4) Solubility; Soluble in lower alcohol, chloroform and ethyl acetate 5) Rf value by silica gel thin layer chromatography;
It shows 0.55 in a developing solvent of n-budanol: acetic acid: water (10: 1: 2) 6) Infrared absorption spectrum (KB _r- disK method); νmax 3400, 3240, 2950, 2920, 2850, 1630, 1580,
1470, 1450,1410, 1385, 1365, 1340, 1160, 820, 720
cm ^-1 7) hydrogen nuclear magnetic resonance spectrum _{(200MHz, CD 3 OD);} δ2.75 (dd, J = 7.08, 7.57, 2H) 1.10~1.65 (m, 21H) 0.87 (d, J = 6.6Hz, 6H ) 8) Color reaction; phosphomolybdic acid-sulfuric acid, ninhydrin positive

B) NK148198B 1) Appearance; white wax 2) Molecular weight: HR-FAMS (m / z): 228.2710
3 (M + H) ⁺ 3) rational formula; C ₁₅ H ₃₃ N 4) solubility; soluble in lower alcohol, chloroform, ethyl acetate 5) Rf value by silica gel thin layer chromatography;
It shows 0.55 in a developing solvent of n-butanol: acetic acid: water (10: 1: 2) 6) Infrared absorption spectrum (KB _r- disK method); νmax 3400 (sh), 3330, 2960, 2920, 2850 , 1635, 15
70, 1480, 1470, 1440, 1390, 1330, 1130, 820, 720 c
m ^-1 7) Hydrogen nuclear magnetic resonance spectrum (200MHz, CD ₃ OD); δ2.73 (dd, J = 7.05, 7.57, 2H) 1.10 ~ 1.65 (m, 23H) 0.83 ~ 0.92 (m, 6H) 8) Color reaction: Positive with phosphomolybdic acid-sulfuric acid and ninhydrin.

The NK148198 of the present invention is as described below.
It is expected as a cancer drug. As a formulation and administration method when used as a pharmaceutical, various conventionally known methods can be applied. That is, injection methods such as injection, oral administration and rectal administration are possible. The dosage form may be injections, powders, granules, tablets, suppositories, and the like.

Various auxiliaries used in medicine, that is, carriers and other auxiliaries such as stabilizers, preservatives, soothing agents, emulsifiers, etc., are required unless adversely affecting NK148198 during formulation. Can be used according to In the preparation, the content of NK148198 can be widely varied depending on the form of preparation, etc.
98 is 0.01 to 100% (weight), preferably 0.1
˜70% (by weight), the rest consisting of carriers and other auxiliaries usually used for medicinal purposes.

The dose of NK148198 varies depending on the symptoms and the like, but is about 0.01 to 800 mg per adult per day. When continuous casting is required, it is preferable to reduce the daily usage amount.

[0023]

[Action]

1. Cell growth inhibitory activity Human ovarian cancer cell line A2780 cells were used as cells.
A2780 cells were cultured at 37 ° C. under 5% CO ₂ using RPMI640 medium supplemented with 10% fetal bovine serum. The cells were seeded on a 96-well plate and cultured for 1 day, and then NK148198A and NK148198B were added respectively. The drug treatment was carried out for 3 days, and the degree of proliferation was evaluated by the MTT method. The results are shown in Table 2.

[0024]

Table 2 Table 2 Cell growth inhibitory activity of NK148198 ──────────────────── Compound IC ₅₀ [μg / ml) ─────────── ────────── A 8.8 B 7.9 ────────────────────

As is clear from this table, the NK of the present invention
148198A and NK148198B have cytostatic activity.

[0026]

As is clear from the above results, NK148198A and NK148198B of the present invention can be expected as active ingredients of novel anticancer agents. Examples of the present invention will be shown below, but these are merely examples and do not limit the present invention in any way, and various modifications are possible.

[0027]

【Example】

Example 1 (1) Fermentation 100 ml of a medium having the following composition was poured into a 500-ml triangular Kolben and sterilized by autoclaving at 120 ° C. for 20 minutes. In addition to this, NK148198 strain (1)
No. 3962) platinum loop was inoculated and cultured at 27 ° C. for 200 days / minute for 2 days, and this was used as a seed mother.

Medium: Starch (Soluble Starch Kanto Kagaku) 2.0% After warm dissolution with an appropriate amount of water, the following components are added. Glucose 0.1% Peptone (MERCK) 0.3% Yeast ext (DIFCO) 0.5% Meat ext (Far East Ehrlich) 0.3% CaCO ₃ 0.2% pH = uncorrected

The seed mother thus obtained was treated with the same medium as 10
Zero 500 ml Kolben was aseptically inoculated so that the inoculum would be 1%, and cultured under the same conditions for 4 days. From about 10 liters of the culture solution thus obtained, filtration was performed to obtain bacterial cells.

(2) Purification 3 L of methanol was added to the cells, and the mixture was stirred for 2 hours and then filtered to obtain a cell extract. Then, 36.2 g of the residue obtained by concentration to dryness under reduced pressure was dissolved in 500 ml of water to adjust the pH to 8.0, and then extracted twice with 500 ml of butanol. This was concentrated under reduced pressure to dryness, and 5.7 g of the resulting residue was mixed with chloroform: methanol = 4: 1.
Then, the mixture was applied to a silica gel column (600 ml) filled in, and developed stepwise with a solvent system of chloroform: methanol = 4: 1, chloroform: methanol = 1: 1, MeOH. The active fraction was concentrated to dryness under reduced pressure, and 448 mg of the resulting residue was charged with methanol to obtain LH-2.
It was applied to a 0 Sephadex (300 ml) column. Next, this active fraction was concentrated to dryness under reduced pressure to obtain 122.6 m.
g was applied to a silica gel column (20 ml) with butanol: water = 40: 1. The active fraction was concentrated under reduced pressure to dryness, and 6
2.7 mg of standard was obtained.

Next, this preparation was dissolved in 10 ml of methanol, and then 100 μl of triethylamine and 144 mg of paramethoxybenzyl S-4,6-dimethylpyrimidin-2-ylthiocarbonate dissolved in 3 ml of dioxane were added. The mixture was stirred at room temperature for 2.5 hours.
After completion of the reaction, methanol was distilled off under reduced pressure, distilled water was added to this, and the mixture was extracted with ethyl acetate. The organic layer was dehydrated with anhydrous sodium sulfate, concentrated under reduced pressure to dryness, and further subjected to silica gel chromatography (10 ml) (hexane: ethyl acetate: 5 :).
Purified in 1), PMZ 99 mg of NK148198
Got

The obtained PMZ body was subjected to reverse phase HPLC (Wa
kosil-II5C18, ψ20mm × 250mm, methanol-H ₂ O (8
7:13), PMZ body 29 of NK148198A
and 67 mg of PMZ body of NK148198B were obtained. Methanol-6N hydrochloric acid (3:
1) It was dissolved in 1 ml and stirred at room temperature for 18 hours. After completion of the reaction, the solvent was distilled off under reduced pressure and neutralized with an aqueous sodium hydroxide solution. After further reduced pressure and concentration to dryness, each was purified by an LH-20 Sephadex column (300 ml) filled with methanol, and then dried under reduced pressure to obtain NK.
148198A15 mg and NK148198B35m
g respectively. These purified NK148198
Infrared absorption spectrum and hydrogen nuclear magnetic resonance spectrum were measured using A and NK148198B. These spectra are as shown in FIGS.

Example 2 NK148198-hydrochloride 30 parts by weight, crystalline lactose 120
30 parts by weight, crystalline cellulose (147 parts by weight) and magnesium stearate (3 parts by weight) were tabletted using a V-type mixer to give 30 tablets.
0 mg tablets were obtained.

[Brief description of drawings]

FIG. 1 shows an infrared absorption spectrum of NK148198A measured by a KBr-Disk method.

FIG. 2 shows a hydrogen nuclear magnetic resonance spectrum of NK148198A measured in deuterated methanol.

FIG. 3 shows an infrared absorption spectrum of NK148198B measured by a KBr-Disk method.

FIG. 4 shows a hydrogen nuclear magnetic resonance spectrum of NK148198B measured in deuterated methanol.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12R 1: 465)

Claims (4)

[Claims] 1. A physiologically active substance N having the following physicochemical properties.
K148198A and NK148198B or a pharmacologically acceptable salt thereof a) NK148198A 1) Appearance; white wax 2) Molecular weight; HR-FAMS (m / z): 214.2532
4 (M + H) ⁺ 3) Rational formula; C ₁₄ H ₃₁ N 4) Solubility; Soluble in lower alcohol, chloroform and ethyl acetate 5) Rf value by silica gel thin layer chromatography;
Shows 0.55 in a developing solvent of n-butanol: acetic acid: water (10: 1: 2) 6) Infrared absorption spectrum (KB _r- disK method); νmax 3400, 3240, 2950, 2920, 2850, 1630, 1580,
1470, 1450,1410, 1385, 1365, 1340, 1160, 820, 720
cm ^-1 7) hydrogen nuclear magnetic resonance spectrum _{(200MHz, CD 3 OD);} δ2.75 (dd, J = 7.08, 7.57, 2H) 1.10~1.65 (m, 21H) 0.87 (d, J = 6.6Hz, 6H ) 8) Black reaction; phosphomolybdic acid-sulfuric acid, ninhydrin positive b) NK148198B 1) Appearance; white wax 2) Molecular weight: HR-FAMS (m / z): 228.2710
3 (M + H) ⁺ 3) rational formula; C ₁₅ H ₃₃ N 4) solubility; soluble in lower alcohol, chloroform, ethyl acetate 5) Rf value by silica gel thin layer chromatography;
Shows 0.55 in developing solvent of n-butanol: acetic acid: water (10: 1: 2) 6) Infrared absorption spectrum (KB _r- disK method); νmax 3400 (sh), 3330, 2960, 2920, 2850, 1635 , 15
70, 1480, 1470, 1440, 1390, 1330, 1130, 820, 720 c
m ^-1 7) Hydrogen nuclear magnetic resonance spectrum (200MHz, CD ₃ OD); δ2.73 (dd, J = 7.05, 7.57, 2H) 1.10 ~ 1.65 (m, 23H) 0.83 ~ 0.92 (m, 6H) 8) Color reaction; phosphomolybdic acid-sulfuric acid, positive for ninhydrin. 2. A microorganism belonging to the genus Streptomyces and having the ability to produce the physiologically active substance NK148198A or NK148198B is cultured in a medium and the physiologically active substance NK148198A or NK148198 is cultured in the culture.
A physiologically active substance NK148198A or NK148198 characterized by producing and accumulating B and collecting it
Manufacturing method of B. 3. An antitumor agent comprising a physiologically active substance NK148198A or NK148198B and a pharmacologically acceptable salt thereof as an active ingredient. 4. A physiologically active substance NK148198A or N
Streptomyces sp. NK148198 strain having the ability to produce K148198B.